Human cytomegalovirus (HCMV) causes considerable morbidity and mortality in newborns and immunocompromised patients and may have subtle but widespread health effects in the large fraction of humans who have life-long quiescent infections. Like many other viruses, HCMV produces proteins that ensure its survival in the face of myriad antiviral defense systems present in host cells. Understanding how these critical viral proteins such as the double-stranded RNA-binding protein hTRS1 function will be useful understanding the viral life cycle and may enable identification of new targets for antivira drugs and development of vaccines to prevent HCMV disease. This proposal aims to elucidate why hTRS1 is essential for the virus, how it functions, and how it has adapted to the changes in the cellular protein kinase R (PKR) pathway that have occurred repeatedly during primate evolution. hTRS1 is known to interact with several viral and cellular proteins and to inhibit PKR, other double-stranded RNA-activated pathways, autophagy and possibly others host defenses. Analyses of specific hTRS1 mutants that retain or lack defined interaction regions will enable determination of which functions of hTRS1 are critical for HCMV replication. Comparative analyses of hTRS1 and its primate cytomegalovirus homologs will provide perspective on which of the functions and mechanisms of the ancestral TRS1 gene have been conserved during evolution. Intriguingly, rhesus CMV (RhCMV) TRS1 functions to evade PKR in some Old World monkey cells but not others, possibly due to polymorphisms in the PKR genes. Genetic manipulations of the PKR gene in monkey cells coupled with experimental evolution of recombinant viruses containing weak TRS1 variants will reveal how changes in PKR alter its sensitivity to TRS1 and, conversely, how TRS1 can adapt to overcome otherwise resistant PKR alleles. In addition to aiding the development of the animal models, these studies will provide insights into the fundamental roles and evolution of dsRNA-binding proteins found in many viral systems.